CN110760521A - Transcription factor NAC1 for improving expression of wheat storage protein gene and application thereof - Google Patents
Transcription factor NAC1 for improving expression of wheat storage protein gene and application thereof Download PDFInfo
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Abstract
The invention discloses a transcription factor capable of improving the gene expression of wheat grain storage protein (SSP)NAC1And uses thereof, the transcription factor isTuNAC1And anTuNAC1Homologous genes in common wheatTaNAC1Three copies of (a):TaNAC‑A1、TaNAC‑B1orTaNAC‑D1Which isThe coding region consists of nucleotide sequences shown by SEQ ID No.1, SEQ ID No.2, SEQ ID No.3 and SEQ ID No.4 respectively. In the present invention, the wheat is from Ural chart (Triticum urartu) Is/are as followsTuNAC1Overexpression in arabidopsis significantly increased the transcription of the SSP gene in immature seeds and the accumulation of SSP in mature seeds. Silencing in common wheatTaNAC1Resulting in a significant reduction in SSP content in mature seeds of the RNAi line.
Description
Technical Field
The invention relates to the field of genetic engineering, in particular to a transcription factor for improving the expression of a wheat storage protein gene and application thereof.
Background
Common wheat (A), (B), (C), (Triticum aestivumL, 2n =6x =42, AABBDD) is one of the major food crops for humans. Wheat plays a great role in human history and society, and flour can be processed into various gourmets because of the viscoelastic property of dough given by the storage protein (SSP) in grains.
Both the genetic composition and protein content of the storage protein can affect the flour processing quality of wheat. The expression of the SSP gene is mainly regulated at the transcription level, so that the regulation of the expression rate of the SSP gene in the grain filling process is the key for changing the final accumulation amount of the SSP in endosperm. Transcription factors regulate the expression of the SSP gene by binding to cis-acting elements in the promoter region of the SSP gene. To date, three major classes of transcription factors, bZIP, Dof and R2R3 MYB, have been identified in cereal crops as being involved in SSP gene expression regulation. These three transcription factors bind to the cis-acting elements GLM, P-box and 5 '-AACNNA-3' of the SSP gene promoter region, respectively. Because of its large genome, bZIP transcription factors regulating SSP gene expression have been identified in wheat by homologous cloning onlySPAAnd Dof-like transcription factorsWPBFAnd R2R3 MYB transcription factorTaGAMybBut their regulation mechanism is less studied. However, the promoter region of SSP gene contains a large number of cis-acting elements combined with other transcription factors except bZIP, Dof and R2R3 MYB, etc., and the transcription factor without the corresponding cis-acting element in the promoter region of SSP gene can participate in the expression control of SSP gene through the interaction between the transcription factors, which suggests that there are more transcription factorsThe identification of the new transcription factors greatly enriches the gene resources for improving the wheat quality.
Ural chart wheat (Triticum urartu2n =2x =14, AA) is the a genome donor of triticum aestivum, whose whole-gene sequencing has been completed. In the genome of wheat, Ural chart, 888 transcription factors are predicted. In view of the similar expression pattern between the transcription factor and the target gene regulated by the transcription factor, the transcriptome of the endosperm of the wheat in the grain filling stage of the Ural chart can be analyzed by RNA-Seq to find the transcription factor with the similar expression pattern with the SSP gene, and the transcription factors can be candidate genes for regulating the SSP gene expression.
Disclosure of Invention
The transcriptome sequencing data analysis of endosperm of kernel filling stage of wheat G1812 of the Ular pattern shows that the transcripts of 22 SSP genes expressed by the wheat G1812 of the Ular pattern start 5 days after flowers and reach a peak at 10-15 days after flowers and then rapidly decline. Through co-expression analysis identifyTuNAC1Has similar expression pattern with SSP gene. Co-transformation of both wheat protoplast cells and endosperm indicatesTuNAC1Promoting the activity of SSP gene promoter. TuNAC1 subcellular localization to the nucleus, it bound to the cis-acting element 5 '-TTGTGTGTTG-3' of the SSP gene promoter region. The overexpression experiment of endosperm and the detection of a dual-luciferase reporter gene system prove thatTuNAC1Homologous genes in common wheatTaNAC1Can up-regulate SSP gene expression, andTaNAC-B1the intensity of the copy is maximal. The results of Arabidopsis transgenesis also showTuNAC1Up-regulate the expression of all SSP genes in immature seeds and ultimately increase the content of mature seed storage proteins. In contrast, inhibition in common wheatTaNAC1Finally reducing the storage protein content in mature seeds of the RNAi strain
The invention aims to provide a transcription factor for regulating SSP gene expression, and the transcription factor can up-regulate SSP gene expression.
The transcription factor provided by the inventionTuNAC1Derived from Wulare map wheat and has the nucleotide sequence composition shown in SEQ ID No.1The gene of (1). The DNA shown in SEQ ID No.1 has the length of 1071 bp, belongs to NAC family and codes protein of 356 amino acid residues.TuNAC1The homologous gene in common wheat isTaNAC1It has three copiesTaNAC-A1AndTaNAC-B1andTaNAC-D1they are genes consisting of nucleotide sequences shown by SEQ ID No.2, SEQ ID No.3 and SEQ ID No.4 respectively. WhereinTaNAC-A1AndTaNAC-D1the length of the DNA sequence of (1) is 1071 bp, and the DNA sequence encodes 356 amino acid residues of protein;TaNAC-B1the DNA sequence of (1) has a length of 1074 bp and encodes a protein of 357 amino acid residues.
Also, the present invention provides amplificationTuNAC1AndTaNAC1the sequence of the forward primer is shown as SEQ ID No.5, and the sequence of the reverse primer is shown as SEQ ID No. 6.
The invention also provides a specific monoclonal antibody which hybridizes to TaNAC1 in vivo and is prepared from the peptide fragment sequence shown in SEQ ID No. 7.
The invention also provides the transcription factorTuNAC1An expression vector.
The invention provides a method for determining and regulating candidate transcription factors expressed by SSP genes by using co-expression analysis, which finds the transcription factors with similar expression patterns to the SSP genes by analyzing transcriptome of endosperm of wheat in the filling stage of Ural chart through RNA-Seq.
The invention provides a method for rapidly detecting a transcription factor regulation and control SSP gene, which co-transforms an overexpression vector of the transcription factor and a report vector of driving GFP expression by an SSP gene promoter into a wheat protoplast cell.
The invention provides a method for detecting the regulation and control strength of a transcription factor on an SSP gene promoter region, wherein an over-expression vector of the transcription factor and a report vector for driving Firefly luciferase (Firefly luciferase) gene expression by an SSP gene promoter are co-transformed into an arabidopsis thaliana protoplast cell, and Renilla luciferase (Renilla luciferase) is used as a control to detect the strength of the transcription factor for regulating the SSP gene promoter activity.
The present invention provides a method for detecting the regulation of SSP gene expression by transcription factors through endosperm overexpression, which will be describedThe transcription factorTuNAC1The overexpression vector is used for transforming endosperm 15 days after common wheat blossoming by a gene gun method, dark culturing is carried out in a hypertonic culture medium for 48 hours, and the change of SSP gene expression in the endosperm is detected by RT-PCR.
The invention also provides a transcription factor for prokaryotic expressionTuNAC1The method of (1), which comprises subjecting the transcription factor toTuNAC1Connecting and integrating into a prokaryotic expression vector, and transforming into a prokaryotic cell; the expression vector is pGEX-4T-1 vector, and the prokaryotic cell is escherichia coli.
The invention also provides the transcription factorTuNAC1Use in plants to increase SSP gene expression: the transcription factor isTuNAC1Over-expression in plants and screeningTuNAC1An overexpressed transgenic positive plant.
The application of the plant is Arabidopsis thaliana which contains the transcription factorTuNAC1Transforming Arabidopsis thaliana with the overexpression vector of (4)TuNAC1Overexpression of the gene.
The invention also provides the transcription factorTaNAC1Use in plants to inhibit SSP gene expression: transcription factors described in plantsTaNAC1Silencing and screening outTaNAC1Transgenic positive plants with the gene suppressed and expressed.
The plant is common wheat, which is obtained by containing the transcription factorTaNAC1The RNAi vector is used for transforming common wheat to realizeTaNAC1Silencing of the gene.
The invention provides detectionTaNAC1Primer pairs for positive RNAi transgenic lines characterized by: the sequence of the forward primer is shown as SEQ ID No.8, and the sequence of the reverse primer is shown as SEQ ID No. 9.
The invention provides a method for screening TuNAC1 combined cis-acting elements in vitro, which divides a promoter region of an SSP gene into 10 fragments, wherein the length of each fragment is about 50 bp, the adjacent two fragments are overlapped by about 10 bp, the fragments are made into probes by using biotin labels, the probes and TuNAC1 recombinant protein are incubated together, and the combination of TuNAC1 and the probes is detected by a gel retardation experiment.
The invention providesSupplied Ural pattern wheatTuNAC1Up-regulating the expression of all SSP genes in the immature seeds of transgenic Arabidopsis thaliana and finally increasing the content of storage proteins in the mature seeds. Its homologous gene in common wheatTaNAC1Binds in the endosperm to the promoter region of the SSP gene, thereby regulating expression of the SSP gene. Overexpression in endospermTaNAC1Can significantly up-regulate the transcription of SSP gene. At the same time, inhibition in common wheatTaNAC1Can finally obviously reduce the content of the storage protein in the mature seeds of the RNAi strain. Therefore, the temperature of the molten metal is controlled,NAC1has important significance for improving the quality of wheat.
Drawings
FIG. 1 wheatNAC1Has similar expression pattern with SSP gene in endosperm of wheat filling stage. Wherein A is Ular pattern wheat in RNA-Seq analysisTuNAC1Clustering with expression patterns of the SSP gene; b is RT-PCR result to prove that the Wulare pattern wheatTuNAC1The expression pattern is similar to that of the endosperm of the SSP gene in the grouting period; the result that C is RT-PCR proves that the common wheat is in Chinese springTaNAC1Three copies ofTaNAC-A1、TaNAC-B1AndTaNAC-D1has similar expression pattern with the endosperm of SSP gene in the grouting period.
FIG. 2 wheatNAC1The gene is unique to wheat family and has highest expression in endosperm. Wherein A isNAC1And the homologous genes of the wheat family are independently gathered into a branch in the analysis of the evolutionary tree; b isTaNAC1Has higher expression level in endosperm 20 d after florescence than other tissues.
FIG. 3 TuNAC1 is localized to the nucleus and has transcriptional activation activity at its C-terminus. Wherein, a and B show that TuNAC1 localizes to the nucleus in both arabidopsis (a) and wheat (B) leaf protoplast cells; c is obtained by predictionTuNAC1A functional region of (a); d is yeast double hybrid and shows that the C-terminal (177-753 bp) of TuNAC1 has transcriptional activation activity.
FIG. 4 wheatNAC1Improve the transcription activity of SSP gene promoter area and finally improve the expression of SSP gene. Wherein A is the result of wheat protoplast cotransformationTuNAC1Increasing the promoter activity of the SSP gene; b isTuNAC1Increasing the promoter activity of the SSP gene in the endosperm of wheat; c isTuNAC1Increasing the promoter activity of the SSP gene in a dual-luciferase reporter gene system; d isTaNAC1Overexpression in the endosperm increases transcription of the SSP gene; e isTaNAC1The promoter activity of the SSP gene is increased in a dual-luciferase reporter gene system.
FIG. 5 TuNAC1 binds to cis-acting element 5 '-TTGTGTTG-3' in vitro. Wherein A isTuNAC1Prokaryotic expression of (3); b and C are TuNAC1 for high molecular weight glutenin gene in gel migration assayTuGlu-1AxAndTuGlu- 1Aybinding of promoter fragments; d is a promoter fragment bound by TuNAC1 and has the same motif; e is verification of TuNAC1 binding to cis-acting element 5 '-TTGTGTTG-3' by point mutation; f is the confirmation of TuNAC1 binding to the cis-acting element 5 '-TTGTGTTG-3' by probe competition.
FIG. 6 TaNAC1 binds to the promoter of the SSP gene in vivo. Wherein, A and B are TaNAC1 antibody which can be specifically combined with protein with the same molecular weight and size with TaNAC1 in Westernblot; the antibody of TaNAC1 binds TaNAC1 in the co-immunoprecipitation at C, and the promoter region of SSP gene is enriched by ChIP-PCR.
FIG. 7TuNAC1The content of storage protein in the transgenic arabidopsis seeds is improved. Wherein A isTuNAC1Overexpression in transgenic Arabidopsis positive lines; b isTuNAC1Improving the expression of the storage protein gene in the immature seed of the transgenic arabidopsis; c is a standard curve for measuring the content of the transgenic arabidopsis seed storage protein; d isTuNAC1Improving the content of storage protein in the mature seeds of the transgenic arabidopsis; e is SDS-PAGE displayTuNAC1The content of each subunit of the storage protein in the mature seeds of the transgenic arabidopsis is improved.
FIG. 8TaNAC1The storage protein content in the mature seeds of the RNAi strain is significantly reduced. Wherein A isTaNAC1The Glutenin (Glutenin) content in mature seeds of the RNAi strain is obviously reduced compared with that of a wild type; b isTaNAC1The alcohol soluble protein (Gliadin) content in mature seeds of the RNAi strain is obviously reduced compared with that of a wild type; c isTaNAC1Mature seed of RNAi strainThe Total storage protein content (Total SSPs) in the seed is significantly reduced compared to the wild type; d isTaNAC1The bands of each storage protein in the mature seeds of the RNAi strain were stained less strongly on SDS-PAGE gel than in the wild type.
Detailed Description
The experimental procedures used in the following examples are all conventional procedures unless otherwise specified. Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.
Example 1 Ural wheatTuNAC1And its homologous gene in common wheatTaNAC1Obtained by
1. Ural pattern wheatTuNAC1And SSP gene co-expression analysis, which comprises the following steps:
(1) RNA-Seq analysis: selecting small ears in the middle of the ears of the Wulare diagram wheat G1812 which has been subjected to genome sequencing, stripping seeds from glumes, cutting off embryos, collecting 10 endosperm of the seeds in a mortar frozen by liquid nitrogen, adding the liquid nitrogen, and grinding the endosperm into powder; extracting total RNA by guanidine hydrochloride method, and performing primary purification and recovery by using RNeasy Plant Mini Kit (Qiagen, Hilden, Germany); the concentration and quality of RNA are detected by a ultramicro spectrophotometer NanoDrop 2000, and are verified by 1.0% agarose gel electrophoresis, so that each sample has at least 5 mug and the concentration is over 100 ng/muL. mRNA was further purified using the DynabeadsmRNA Purification Kit (Invitrogen, Carlsbad, Calif., USA) and was subjected to library construction and sequencing by the sequencer.
(2) Calculation of Gene expression level (RPKM): the Raw data filters low-quality reads to obtain clean reads; adding the sequence of the coding region of the cloned SSP gene into the genome sequence of G1812 to form an integrated genome sequence, and ensuring that each SSP gene only retains one sequence; aligning clean reads to a reference genome integrated by G1812 by using TopHat (TopHat v2.0.10) software, and reserving reads aligned to a unique position on the genome; the read count for each gene was calculated by HTseq (Version 0.5.4p5), rpkm (reads per genomic mapped reads) which is the number of reads aligned to the gene divided by the number of all reads aligned to the genome and the length of RNA.
(3) Co-expression analysis: the expression patterns of 888 transcription factors and the SSP gene which are predicted from the wheat genome sequence of the Ural chart are clustered together, so that the transcription factors which have similar expression patterns with the SSP gene are found out and are used as candidate genes participating in the regulation and control of the SSP gene expression. As shown in FIG. 1A, the SSP gene of Ular charm wheat was specifically expressed in endosperm, beginning at 5d after anthesis, then rapidly increased and reached a peak at 10 d to 15 d after anthesis, and then rapidly decreased until 20 d after anthesis reached a minimum. Among all the transcription factors, the transcription factor,TuNAC1(TRIUR3_25137) Most similar to the expression pattern of SSP gene, they are also in one branch of cluster analysis,TuNAC1the correlation coefficient with the expression pattern of the SSP gene reaches 0.95.
(4) Wheat (Triticum aestivum L.)NAC1Cloning of the genes: according toTRIUR3_25137The 5 'and 3' terminal of (3) and designed amplificationTuNAC1The full-length primer (the sequence of the forward primer is shown as SEQ ID No.5, the sequence of the reverse primer is shown as SEQ ID No. 6), the mRNA of 15 d endosperm after the wheat florescence of the Ural chart is used as a template, and the mRNA is obtained by cloningTuNAC1The sequence is SEQ ID No. 1. In addition, total RNA of endosperm 15 d after spring flower of common wheat is extracted by guanidine hydrochloride method and is reversely transcribed into cDNA, and the total RNA is utilizedTuNAC1The full-length primer is amplified to obtain common wheatTaNAC1Three copies ofTaNAC-A1,TaNAC-B1AndTaNAC-D1and the sequences are SEQ ID No.2, SEQ ID No.3 and SEQ ID No.4 respectively.
(5) Wheat (Triticum aestivum L.)NAC1Verification of gene and SSP gene expression patterns: in the endosperm of the wheat in the filling stage of the Ural chart, the use ofTuNAC1And SSP gene specific primers, and the expression patterns of the two are verified by RT-PCR. The results are shown in figure 1B of the drawings,TuNAC1and expression of the selected SSP gene both started at 5d after anthesis, peaked at 10-15 d after anthesis and subsequently decreased, which is essentially identical to the result of RNA-Seq. In addition, RT-PCR detects the endosperm of the common wheat in the China spring filling stageTaNAC1And SSP gene expression pattern, as shown in FIG. 1C,TaNAC-A1、TaNAC-B1andTaNAC-D1having identity to SSP geneExpression pattern, i.e. starting at postanthesis 5d and rising slowly from postanthesis 5-15 d, then rising rapidly and reaching a peak at postanthesis 20 d, then the expression level starts to decline; this also suggests that these three copies may be involved in regulation of SSP gene expression in Triticum aestivum.
2. Wheat (Triticum aestivum L.)NAC1Evolutionary tree analysis and tissue-specific expression analysis of genes
(1) And (3) analyzing the evolutionary tree: will be provided withTuNAC1AndTaNAC1and the amino acid sequences of the genes with more than 80 percent of sequence similarity in GenBank (before 2017-12) are compared by Clustal Omega (http:// www.ebi.ac.uk/Tools/msa/clustalo /) software, then an evolutionary tree is constructed by Mega 5.05 (http:// www.megasoftware.net /), an adjacent approach (Neighbor-join) and a P-distance model are adopted, and Bootstrap test is carried out by 1,000 times of repetition. The results are shown in figure 2A as,TuNAC1and its homologous genes in the wheat family were clustered as one branch alone in the evolutionary tree analysis, while genes from other families were in different branches, indicating thatNAC1The gene is peculiar to wheat family.
(2) Tissue-specific expression analysis: extracting total RNA of root, stem, flag leaf and endosperm of seedling and post-flowering 15 d plant of common wheat in Chinese spring by guanidine hydrochloride methodTaNAC-A1、TaNAC-B1AndTaNAC-D1the specific primers of (1) were verified for their tissue-specific expression by RT-PCR, and the results are shown in FIG. 2B, at the roots of seedlings and postanthesis 15 d,TaNAC-B1andTaNAC-D1there is a small amount of expression of the gene,TaNAC-A1trace expression is present; in the endosperm 15 d after the flower,TaNAC-A1、TaNAC- B1andTaNAC-D1are abundantly expressed and are expressed inTaNAC-A1The expression level of (2) is highest.
3. Subcellular localization and transcriptional activation Activity assay for TuNAC1
(1) Subcellular localization: will be provided withTuNAC1And pJIT163-hGFP (http:// www.pgreen.ac.uk /) vector A recombinant plasmid pJIT163-TuNAC1-hGFP was constructed for transformation of Arabidopsis thaliana leaf protoplast cells. In addition, the 35S promoter in the pJIT163-TuNAC1-hGFP recombinant plasmid was replaced with the ubiquitin promoter to transform wheat leaf protoplast cells. The results are shown in FIG. 3A and FIG. 3TuNAC1 was shown to localize to the nucleus of protoplast cells. TuNAC1 was also localized to the nucleus in Arabidopsis leaf protoplast cells.TuNAC1Localization in the nucleus indicates that it functions in the nucleus, consistent with the characteristics of transcription factors.
(2) Detection of transcriptional activation Activity: the DNA binding region and the transcriptional activation region of TuNAC1 protein were predicted by NCBI BLAST (https:// BLAST. NCBI. nlm. nih. gov/BLAST) alignment, and as a result, as shown in FIG. 3C, 51-174 bp of TuNAC1 was its DNA binding region (NAM) and 177-753 aa was its transcriptional activation region. And both were cloned separately and ligated with pGBKT7 (Clontech, California, USA) into recombinant plasmids. The recombinant plasmids of the DNA binding and transcriptional activation regions and pGBKT7 were co-transformed with pGADT7 (Clontech, California, USA) to yeast strain AH109 (Clontech, California, USA), respectively. pGBKT7-53 (Clontech, California, USA) and pGADT7 were used in combination as positive controls, and pGBKT7-Lam (Clontech, California, USA) and pGADT7 were used in combination as negative controls. As a result, as shown in FIG. 3D, all strains grew normally on the two-lacking solid medium (SD/-Leu/-Trp), but only the strain transformed with the recombinant plasmid for the complete gene and transcriptional activation region grew normally on the four-lacking solid medium (SD/-Ade/-His/-Leu/-Trp), while the strain transformed with the recombinant plasmid for the DNA binding region could not grow on the four-lacking solid medium. This indicates that TuNAC1 has transcriptional activation activity, and that it is its transcriptional activation region that functions in transcriptional activation activity.
Example 2 wheatNAC1Function of the Gene
1. Wheat (Triticum aestivum L.)NAC1Direct regulation of SSP genes
(1) Protoplast and endosperm cotransformation: the pJIT163-UBI-hGFP vector (ampicillin resistance), supplied by Cabernet Gaussler, institute of genetics and developmental biology, of the Chinese academy of sciences, driven the expression of GFP by the ubiquitin promoter. By usingTuNAC1The coding region of (a) was substituted for the coding region of GFP to construct its overexpression vector pJIT163-UBI-TuNAC1, while the ubiquitin promoter was substituted with the promoter of SSP gene (2,000 bp) to construct the reporter vector pJIT163-SSP promoter-hGFP. Co-transformation of transcription factor over-expression vector and report vectorWheat protoplast cells, i.e. cells which can be examined by the intensity of fluorescence excitationTuNAC1Regulatory activity on SSP gene promoter. The results are shown in figure 4A,TuNAC1can bind to SSP gene promoter to activate GFP expression.
Disinfecting seeds 15 days after spring flowering in China with 70% alcohol, peeling off seed coats in a super clean bench, longitudinally cutting the seeds with a scalpel, placing the seeds in a range of 2-3 cm from the center of a hypertonic culture medium with the cut faces upward, and performing hypertonic treatment for 3-4 h; adding sterilized 50% glycerol to the gold powder to a final concentration of 150 mg/mL; 50 μ L of diluted gold powder was taken, and 3 μ L of plasmid (pJIT163-UBI-TaNAC1, 1 μ g/. mu.L) and 50 μ L of 2.5M CaCl were added2Mixing with 20 μ L of 0.1M spermidine, and blowing and mixing with a pipette; will be provided withTuNAC1The overexpression vector and the reporter vector bombard endosperm together by a gene gun, and the detection can be realized by the strength of fluorescence excitationTuNAC1Regulatory activity on SSP gene promoter. The results are shown in figure 4B which shows,TuNAC1can bind to SSP gene promoter to activate GFP expression.
(2) Dual luciferase reporter gene system detectionNAC1Regulation of the SSP gene promoter: will be provided withTuNAC1、TaNAC-A1、TaNAC-B1AndTaNAC-D1respectively recombined in pRT107 vectors (provided by Zhang Song researchers of institute of genetics and developmental biology, Chinese academy of sciences) to drive 35S promoterTaNAC1And (3) performing mass expression, and replacing a 35S promoter of a reporter gene vector by a promoter (2,000 bp) of an SSP gene to drive the expression of a firefly luciferase (firefly luciferase) gene. The two recombinant vectors are co-transformed into arabidopsis protoplast cells, and Renilla luciferase (Renilla luciferase) is used as a control, so that the wheat can be detectedNAC1Promoter strength to the SSP gene promoter region. Fluorescence intensity was measured using the Dual-Luciferase Reporter Assay System kit (Promega, Madison, USA) and Gloma20/20 Luminometer (Promega, Madison, USA). The results are shown in figures 4C and 4D,TuNAC1the SSP gene is remarkably improvedTuGlu-1Ax,TuGlu-1Ay,TuA3-520,TuA3-538a,Gli-α-8AndGli-γ-1the promoter activity of (1). In thatTaNAC1Of the three copies of the first copy of the second copy,TaNAC-B1for SSP baseThe up-regulation amplitude of the promoter activity is highest; compared to a control transformed with the reporter gene alone,TaNAC-B1can be adjusted up remarkablyGlu-1Bx7、Glu-1Dx2AndGlu-1Dy12while the promoter activity of (a) is significantly up-regulatedGlu-1Bx8、D3-525、D3-578AndGli-α-7the promoter activity of (1).TaNAC-A1AndTaNAC-D1except that it can be adjusted up remarkablyGlu-1Bx7AndGlu-1Dx2the activity of the promoter region has no significant effect on the activity of the promoter region of other SSP genes
（3）TaNAC1Endosperm over-expression of (a): disinfecting seeds 15 days after spring flowering in China with 70% alcohol, peeling off seed coats in a super clean bench, longitudinally cutting the seeds with a scalpel, placing the seeds in a range of 2-3 cm from the center of a hypertonic culture medium with the cut faces upward, and performing hypertonic treatment for 3-4 h; adding sterilized 50% glycerol to the gold powder to a final concentration of 150 mg/mL; 50 μ L of diluted gold powder was taken, and 3 μ L of plasmid (pJIT163-UBI-TaNAC1, 1 μ g/. mu.L) and 50 μ L of 2.5M CaCl were added2Mixing with 20 μ L of 0.1M spermidine, and blowing and mixing with a pipette; bombarding endosperm with a gene gun, and then culturing in a hypertonic culture medium for 24 hours in a dark place; extracting total RNA of endosperm, and performing reverse transcription to obtain cDNA; RT-PCR validationTaNAC-A1、TaNAC-B1AndTaNAC-D1the transient expression of (A) and its effect on SSP gene expression, as shown in FIG. 4E, compared with the wild-type endosperm into which no over-expression plasmid was introduced,TaNAC-A1、TaNAC-B1andTaNAC-D1can up-regulate SSP gene expression, among othersTaNAC-B1The strength of the up-regulation of (a) is maximum.
2. Combination of wheat NAC1 and SSP gene promoter
(1) Gel blocking screen for cis-acting elements bound by TuNAC 1: will be provided withTuNAC1Recombinant in pGEX-4T-1 vector (GEHealthcare, Chicago, USA), TuNAC1 recombinant protein fused with GST tag was expressed and purified by the present inventors as shown in FIG. 5ATuNAC1The recombinant protein of (1). Dividing the HMW-GS gene promoter region from 0 to-500 bp into 10 segments with the length of about 60bp, and overlapping the adjacent two segments by about 10 bp. The probe was synthesized by Shanghai Jun corporation, and the biotin label was added to the 5' end of the probe, only one strand of the probe was labeled. After probe synthesis, ddH was used2O is diluted to 100. mu.M, 1. mu.L each of the sense strand and the antisense strand is taken and mixed in a 200. mu.L PCR tube, and double strands are synthesized by annealing in a PCR instrument. The temperature was first reduced to 95 ℃ for 5 min and then to 25 ℃ at a rate of 0.6% (about 1.5 h). Double-stranded probes were diluted to 2 pM of working solution. The results are shown in FIGS. 5B, 5C and 5D for TuNAC1 andTuGlu-1Axbinding of C4 and C5 probes to the promoter region simultaneouslyTuGlu-1AyThe C8 probe in the promoter region binds. Alignment of the sequences of these three probes revealed that they had the overlapping motif 5 '-ttgtgtgttg-3', presumably the cis-acting element to which TuNAC1 binds in the SSP gene promoter region. Point mutations to 5 '-TTGTGTTG-3' (fig. 5E) and competitive experiments (fig. 5F) also confirmed that TuNAC1 binds to it.
(2) ChIP-Seq verified the binding of TaNAC1 and the SSP promoter: monoclonal antibodies were prepared using peptide portion FGNHHYQNMATPPR of TaNAC1 (SEQ ID No. 7), and as shown in fig. 6A, 6B and 6C, antibodies that specifically bind TaNAC1 in Western blot and co-immunoprecipitation were obtained. The antibody is used for performing ChIP-Seq on the endosperm of 20 d after China spring flowering of common wheat by the same method as Li et al, 2015. In the analysis of ChIP-Seq data, we used Chinese spring gene sequence (ftp:// ftp. ensimblegenes. org/pub/plants/release-36/fasta/tritium _ aestivum/dna /) as reference genome and added SSP gene sequence cloned from Chinese spring. The results are shown in Table 1, HMW-GS Gene: (Glu-1AxNull、Glu-1Ay、Glu-1By8、Glu-1Dx2AndGlu-1Dy12) And LMW-GS Gene: (A3-502a、D3-575AndD3-591) And prolamin gene (a)Gli-ω-9AndGli-ω-10) The promoter fragment is enriched, and proves thatTaNAC1Binds to the promoter region of the SSP gene in vivo. Also, the results of ChIP-PCR (FIG. 6D) confirmed this.
Table 1 the promoter region of the SSP gene was enriched in the ChIP-Seq experiment of TaNAC 1.
| Name | SSP type | Gene |
| Peak_1 | HMW-GS | Glu-1AxNull (pseudogene) |
| Peak_2 | HMW-GS | Glu-1Ay (pseudogene) |
| Peak_3 | HMW-GS | Glu-1By8 |
| Peak_4 | HMW-GS | Glu-1By8 |
| Peak_5 | HMW-GS | Glu-1Dx2 |
| Peak_6 | HMW-GS | Glu-1Dy12 |
| Peak_7 | LMW-GS | A3-502a (pseudogene) |
| Peak_8 | LMW-GS | A3-502a (pseudogene) |
| Peak_9 | LMW-GS | D3-575 |
| Peak_10 | LMW-GS | D3-591 (pseudogene) |
| Peak_11 | Gliadin | Gli-ω-10 |
| Peak_12 | Gliadin | Gli-ω-9 (pseudogene) |
| Peak_13 | Gliadin | Gli-ω-9 (pseudogene) |
3. Transformation and selection of Arabidopsis thaliana
(1) And (3) culturing an arabidopsis plant: arabidopsis thaliana (Arabidopsis thaliana) Columbia ecotype was used for genetic transformation. The seeds are disinfected by mixed liquor of 0.5 percent of sodium hypochlorite and 0.01 percent of Triton X-100, washed by sterilized water, dibbled on an MS culture medium, placed on a greenhouse culture shelf at 4 ℃, kept away from light for 72 hours, transplanted into sterilized nutrient soil (vermiculite: nutrient soil =1:1) after the MS culture medium grows into strong seedlings (about 7 days). The growth conditions were 23 ℃, 60% humidity and 12 h light. Will be provided withTuNAC1Constructed in the pEarleyGate 103 vector (ThermoFisher, Massachusetts, USA), Arabidopsis thaliana Columbia wild type was transformed using the dipping flower method.
(2) And (3) agrobacterium culture: inoculating single colony in 5 ml YEB (Kan 50 mg/L and Rif 60 mg/L) culture medium, after overnight culture, inoculating in 200 ml YEB conical flask according to 1:1000, continuing culture for 10-12 h, when bacterial liquid grows to OD600 value of about 1.0, centrifuging 5000 g for 15 min to collect thallus, and suspending thallus with transformation medium to OD600 value of 0.7-0.8. The components of the transformation medium are as follows: 1/2 MS medium, 5% sucrose, 0.03% Silwet L-77, pH 5.7 (KOH adjusted).
(3) Agrobacterium-mediated arabidopsis transformation: arabidopsis thaliana was transformed by the floral dip method (Clough SJ, 1998). Pouring a transformation medium containing agrobacterium into a 200 ml beaker, inverting the arabidopsis thaliana with flower buds thereon, enabling the whole inflorescence to enter the transformation medium, soaking for 30 s, taking out the arabidopsis thaliana, shaking off excessive water beads on leaves, stems and flowers, laying on the side, putting in a clean plastic basin, and covering with a film, keeping the humidity and culturing for 24 h in a dark place. The film was uncovered, and the Arabidopsis thaliana was placed under light, watered thoroughly, and cultured normally. After the transformed plants grow normally, blossom and bear fruits, and after 2-3 weeks, seeds can be harvested when the siliques are completely withered and yellow and are about to crack. The seeds are stored in 2 ml centrifuge tubes and stored for a short time at normal temperature or for a long time at-20 ℃.
(4) Screening transgenic plants of arabidopsis thaliana: mixing T with disinfectant (0.50% Sodium Hypochlorite, 0.01% Triton X-100)0Seed generation treatment for 15 min, ddH2O flushingCleaning, and standing at 4 ℃ for 3 d; sowing seeds on MS screening culture medium (30.00 mg/L Bialaphos), culturing (23 deg.C, 60% humidity, 12 h light/12 h dark) for 10 d, transplanting normal growth plants into soil until T knot1Seed generation; will T1The generation seeds are individually sowed on an MS screening culture medium (30.00 mg/L Hygromycin), strains with the survival/death ratio of 3:1 are selected, positive strains of the strains are transplanted into soil, and the positive strains are cultivated in a greenhouse (23 ℃, 60 percent of humidity, 12 h of light/12 h of darkness) until T knot occurs2Seed generation; will T2The generation seeds are individually dibbled on an MS screening culture medium (30.00 mg/L Hygromycin), completely survived strains are selected, positive strains of the strains are transplanted into soil, and the positive strains are cultured in a greenhouse (23 ℃, 60 percent of humidity, 12 h of light/12 h of darkness) until T is formed3The seeds of the pure line are generated.
(5) Identification of transgenic plants of Arabidopsis: for the above screened T1Generation, T2The method comprises the steps of substituting an arabidopsis positive plant, extracting a small amount of genome DNA of the arabidopsis positive plant according to a conventional method, taking 1 mul of the genome DNA as a template, carrying out conventional PCR amplification by using a primer pair (shown as SEQ ID No.5 and SEQ ID No. 6), and further detecting the positive plant by using the wild arabidopsis genome DNA as a control. While also being paired with T3And (3) extracting RNA for a small amount of a transgenic plant, carrying out RT-PCR (reverse transcription-polymerase chain reaction) to detect the expression condition of the transgene, and taking the RNA of the wild arabidopsis thaliana as a control. The results are shown in figure 7A,TuNAC1over-expression in transgenic Arabidopsis positive lines.
(6) Phenotype identification of transgenic arabidopsis thaliana: variation in immature seed storage protein gene expression level: method for extracting arabidopsis T by using guanidine hydrochloride method3The purified seed total RNA was reverse-transcribed into cDNA, and expression of SSP gene was detected by RT-PCR using EF1aA4 (AT5G60390) as a control and a primer specific to the Arabidopsis SSP gene (Gao et al, 2016). The results are shown in figure 7B which shows,2S1、2S3、2S4、2S5、CRA1、CRU2andCRU3the expression level in the immature seeds of the three lines is greatly improved compared with that of the wild type.2S2Although the expression level of (2) was not significantly changed in the strain L79, it was found in the strains L8 and L14The expression level of the polypeptide is remarkably improved. In the same way as above, the first and second,CRBthe expression level of (2) is not obviously changed in the strain L14, but the expression level of the strain L8 and the strain L14 is obviously improved. These results all illustrateTuNAC1Up-regulating the expression of the Arabidopsis SSP gene.
(7) Determination of storage protein content of mature seed 15 seeds from wild type and transgenic Arabidopsis thaliana were each placed in 2.0 mL centrifuge tubes and 50. mu.L of Arabidopsis thaliana storage protein extraction buffer [100.00 mM Tris, 0.50% (w/v) SDS, 10.00% (v/v) Glycerol, 20.00% (v/v) β -ME, pH 8.0]Adding steel balls, and breaking by shaking in Geno/Grinder 2000 (OPS Diagnostics LLC, New Jersey, USA) (1,000 rpm, 2 min); after metal bath at 99 ℃ for 5 min, centrifuging at room temperature and 12,000 rpm for 5 min, and obtaining supernatant which is storage protein extracting solution; the concentration of storage proteins was determined with the Quant Kit (GE Healthcare, Buckinghamshire, UK) (fig. 7C). The results are shown in fig. 7D, where the total storage protein content of transgenic arabidopsis strains L8 and L79 is significantly higher than that of the wild type, and the total storage protein content of strain L14 is very significantly higher than that of the wild type, i.e. strain L14 is very significantly higher than that of the wild typeTuNAC1The content of the storage protein in the mature seeds of the transgenic arabidopsis line is improved. In addition, the results of the SDS-PAGE-based assay of the reservoir proteins are shown in FIG. 7E,TuNAC1the content of each subunit of the storage protein in the mature seeds of the transgenic arabidopsis strain is improved.
4. Transformation and screening of common wheat
(1) Constructing RNAi vector and transforming common wheat: will be provided withTaNAC1The positive sequence and the reverse complementary sequence of the conserved region (762 bp-954 bp) of the coding region are recombined in a pUbi-Leal-RNAi vector to constructTaNAC1The RNAi vector of (1). Transforming the transgenic platform of institute of genetics and developmental biology of Chinese academy of sciences by using a gene gun bombardment method to obtain T0Transgenic plants are generated.
（2）TaNAC1Identification and screening of RNAi transgenic lines: converting the obtained T0The transgenic plant is planted in a greenhouse, 2-3 cm leaves are taken, and genome DNA is extracted by a CTAB method. Taking 1 mul of genome DNA as a template, using a primer pair (shown in SEQ ID No.8 and SEQ ID No. 9) to amplify by conventional PCR,and (3) identifying and screening to obtain positive plants by taking wild wheat KN199 genome DNA as a control, and carrying out generation-adding planting in a greenhouse.
（3）TaNAC1Extracting mature seed protein of RNAi transgenic strain: will be provided withTaNAC1T2Seeds of RNAi transgenic lines RNAi # 8, RNAi # 29 and RNAi # 71 are ground into whole powder, and protein extraction and quality index measurement are carried out. Weighing 45mg of whole wheat flour into a2 ml centrifuge tube, adding 1 ml of 70% (v/v) alcohol, shaking for 1 hour at room temperature, centrifuging at 12,000 rpm for 10 minutes, and sucking the supernatant into a new 2 ml centrifuge tube to obtain the prolamin. Adding 1 ml of 7.5% n-propanol (containing 0.3M NaI) into the rest precipitate, mixing uniformly, shaking at room temperature for 30 minutes, centrifuging at 12,000 rpm for 10 minutes, and discarding the supernatant; adding 1 ml of 70% (v/v) alcohol, shaking for 30 minutes at room temperature, centrifuging at 12,000 rpm for 10 minutes, and discarding the supernatant; adding 1 ml of 50% (v/v) isopropanol, incubating at 65 ℃ for 30 minutes while shaking for 2-3 times, centrifuging at 12,000 rpm for 10 minutes, and discarding the supernatant; repeating the above step for 2 times, adding 500 μ l of extract A (50% isopropanol, 0.08M Tris-HCl, pH 8.0 and 1% DTT), incubating at 65 deg.C for 1 hr while shaking for 2-3 times; then 500. mu.l of extract B (50% isopropanol, 0.08M Tris-HCl, pH 8.0 and 1.4% 4-VP) was added, incubated at 65 ℃ for 30 minutes, centrifuged at 12,000 rpm for 10 minutes, and the supernatant was aspirated into a new 2 ml centrifuge tube, which was glutenin.
（4）TaNAC1Determination of mature seed protein of RNAi transgenic strain: the extracted glutenins and gliadins were filtered through a 0.45 μm organic filter, 10 μ l was aspirated for RP-HPLC analysis and quantified using 1 mg/ml BSA standard. As shown in FIGS. 8A, 8B and 8C, the content of glutenins, gliadins and total SSP in the transgenic lines was significantly reduced compared to the control, and the results of SDS-PAGE also showed a significant reduction in the protein band in the transgenic lines (FIG. 8D). These results all indicate inhibitionTaNAC1The expression of (a) can reduce the accumulation of storage proteins in wheat grains.
<110> institute of genetics and developmental biology of Chinese academy of sciences
<120> transcription factor NAC1 for improving expression of wheat storage protein gene and application thereof
<160>9
<210>1
<211>1071
<212>DNA
<213> TuNAC1, Triticum aestivum L)
<400>1
1 ATGGCAGACC ACCTTCAAGT TCAGCGGCAA CAATTAAAGT TCCCTCAGGG GTTCAGGTTT
61 CACCCGACGG ATGTGGAGAT CATCACCTCC TACCTAGTCC CTAAGGTGTT GAACAAAGCA
121 TTCGACCCCA TAGCGGTCGG GGAGGTGGAC TTGAACAAAT GCGAGCCATG GGAACTCCCC
181 GAGAAGGCGA AAATGGGGGA GAAGGAGTGG TACTTCTTCT CCCAAAAGGA CCGCAAGTAC
241 CCCACCGGTA TACGGACCAA CCGAGCCACG ACCGCCGGCT ACTGGAAGGC CACTGGAAAG
301 GACAAGGAGA TCTTCCACCA CGCGACCACA AGTCTCATCG GCATGAAGAA GACGTTGGTC
361 TTCTACAAGG GTAGGGCGCC TAGGGGGGAG AAGACCAACT GGGTCATGCA CGAGTATAGG
421 CTCGAGAGTG GAAAACAAGG AACACCTAGT CTACCCATCG ACATCACCAC CGCCACGGCC
481 ATTAATGCAT CTTCCAAGGA GGAGTATGTG GTTTGCAGGA TATTCCATAA GAGTACTGGA
541 CTCAAGAAGG TAGTGATGTC GTCGTACGCT ATTCCCATGC CAATGTCCAT GGGAGGAGAG
601 GAGCAACATG GCTTCCTTGA ATCCGGTACA TTGCCTGCTT TCATGGGCTA TGGTGCATCA
661 TCATCGCTGG TGCCCCCATC GTCCCTACCT GCATCTTCTT CGTACCAGCT GCATGACACT
721 GGGGCCAGAT CGTCAATGAT GGGTACTGCG GTGCTCCTGA TGATGAACGA CCGCTACTTC
781 GGGAACCACC ACTACCAGAA TATGGCTACC CCACCACGAC CGTTGGTGTC GTTCTACCAC
841 CATGACCACC ACCAGCAGCA GCAACAACAA CAACACATGA TTCAAATGCC GATGCAGATG
901 CAAATAGGTG CGGATGAGGG CCTCATGATC GGGGTCGAGC CTGGGAGCGG GCCATCATCC
961 ATAGTGTCGC AGGAGGATAC TTTGGCCAAG TTGAGCAGCA ACGACGTTGC AACAACTGCT
1021 GAAGAGATCT CATCAGTGAA CATGGGTACG GATGGCATGT GGAAGTACTG A
<210>2
<211>1071
<212>DNA
<213> TaNAC-A1, Triticum aestivum L
<400>2
1 ATGGCCGACC ACCTTCAAGT TCAGCGGCAA CAATTAAAGT TCCCTCAGGG GTTCAGGTTT
61 CACCCGACGG ATGTGGAGAT CATCACCTCC TACCTAGTCC CTAAGGTGTT GAACAAAGCA
121 TTCGACCCCA TAGCGGTCGG GGAGGTGGAC CTGAACAAAT GCGAGCCATG GGAACTCCCC
181 GAGAAGGCGA AAATGGGGGA GAAGGAGTGG TACTTCTTCT CCCAAAAGGA CCGCAAGTAC
241 CCCACCGGTA TACGGACCAA CCGAGCCACG ACCGCCGGCT ACTGGAAGGC CACTGGAAAG
301 GACAAGGAGA TCTTCCACCA CGCGACCACA AGTCTCATCG GCATGAAGAA GACGTTGGTC
361 TTCTACAAGG GTAGGGCGCC TAGGGGGGAG AAGACCAACT GGGTCATGCA CGAGTATAGG
421 CTCGAGAGTG GAAAACAAGG AACACCTAGT CTACCCATCG ACATCACCAC CGCCACGGCC
481 ATTAATGCAT CTTCCAAGGA GGAGTATGTG GTTTGCAGGA TATTCCATAA GAGCACTGGA
541 CTCAAGAAGG TAGTGATGTC GTCGTACGCT ATTCCCATGC CAATGTCCAT GGGAGGAGAG
601 GAGCAACATG GCTTCCTTGA ATCCGGTACA TTGCCTCCTT TCATGGGCTA TGGTGCATCA
661 TCATCGCTGG TGCCCCCATC GTCCCTACCT GCATCTTCTT CGTACCAGCT GCATGACACT
721 GGGGCCAGAT CGTCAATGAT GGGTACTGCG GTGCTCCTGA TGATGAACGA CCGCTACTTC
781 GGGAACCACC ACTACCAGAA TATGGCTACC CCACCACGAC CGTTGGTGTC GTTCTACCAC
841 CATGACCACC AACAGCAGCA GCAACAACAA CAACACATGA TTCAAATGCC GATGCAGATG
901 CAGATAGGTG CAGATGAGGG CCTCATGATC GGGGTCGAGC CTGGGAGCGG GCCATCATCC
961 ATAGTGTCGC AGGAGGATAC TTTGGCCAGG TTGAGCAGCA ACGACGTTGC AACAACTGCT
1021 GAAGAGATCT CATCAGTGAA CATGGGTACG GATGGCATGT GGAAGTACTG A
<210>3
<211>1074
<212>DNA
<213> TaNAC-B1, Triticum aestivum L
<400>3
1 ATGGCAGACC ACCTTCAAGT TCAGCGGCAA CAACTAAAGT TCCCTCAGGG GTTCAGGTTT
61 CACCCGACGG ATGTGGAGAT CATCACCTCC TACCTAGTCC CTAAAGTGTT GAACAAAGCA
121 TTCGACCCCA TAGCGATCGG GGAGGTGGAC CTGAACAAAT GTGAGCCGTG GGAACTCCCC
181 GAGAAGGCGA AAATGGGGGA GAAGGAGTGG TACTTCTTCT CCCAAAAGGA CCGCAAGTAC
241 CCCACCGGTA TACGGACTAA CCGAGCCACG ACTTCCGGCT ACTGGAAGGC CACTGGAAAG
301 GACAAGGAGA TCTTCCACCA TGTGACTACA AGTCTCATCG GCATGAAGAA GACGTTGGTC
361 TTCTACAAGG GTAGGGCGCC AAGGGGGGAG AAGACCAACT GGGTCATGCA TGAGTATAGG
421 CTCGAGTGTG GAAAACAAGG AACACCTGGT CTACCCACCG ACATCACCAC CGCCACAGCC
481 ATTAATGCAT CTTCCAAGGA GGAGTATGTG GTTTGCAGGA TATTCCATAA GAGCACTGGA
541 CTCAAGAAGG TAGTTGTGTC GTCGTACGCC ATTCCCATGT CAATGCCCAT GGGAGCAGAG
601 GAGCAACATG CCTTCCATGA ATCCGGTACA TTACCTCCTT TCATGGGCTA TGGTGCATCA
661 TCATCCCTGG TGCCCCCATC GTCCGTACCT ACATCTTCTT CATACCAGCT GCATGACGTT
721 GGGGCCAGAT CGTCAATGAT GGGTAGTGCG GTGCTCCCGA TGATGAACGA CCACTATTTC
781 AGGAATCACC ACTACCAGAA TATGGCTACC CCACCACGAC CGTTGGTGTC GTTCTACCAC
841 CATGACCACC ACCACCAGCA ACAACAACAA CAACAACACA TGATTCAAAT GCCGATGCAG
901 ATGCAGATAG GTGCAGATGA GGGCCTTATG ATCGGGGTCG AGCCTGGGAG CGGGCCGTCA
961 TCCATAGTGT CGCAGGAGGA CACTTTGGCC AGGTTCAGCA GCAACGACGT TGCAACAACT
1021 GCTGATGAGA TCTCGTCAGT GAACATGGGT ACGGATGACA TATGGAAGTA CTGA
<210>4
<211>1071
<212>DNA
<213> TaNAC-D1, Triticum aestivum L
<400>4
1 ATGGCAGACC ACCTTCAAGT TCAGCGGCAA CAACTAAAGT TCCCTCAGGG GTTTAGGTTT
61 CACCCGACAG ATGTGGAGAT CATCACCTCC TACCTAGTCC CTAAGGTGTT GAACAAAGCA
121 TTCGACCCCA TAGCGGTCGG GGAGGTGGAC CTAAACAAAT GCGAGCCGTG GGAACTCCCC
181 GAGAAGGCGA AAATGGGGGA GAAGGAGTGG TACTTCTTCT CCCAAAAGGA CCGCAAGTAC
241 CCCACCGGTA TACGGACCAA CCGAGCCACG ACCGCCGGCT ACTGGAAGGC CACTGGAAAG
301 GACAAGGAGA TCTTCCACCA CGCGACCACA AGTCTCATCG GCATGAAGAA GACGTTGGTC
361 TTTTACAAGG GTAGGGCGCC CAGGGGGGAC AAGACCAACT GGGTCATGCA CGAGTATAGG
421 CTCGAGAGTG GAAAACAAGG AACACCTAGT CTACCCACCG ACATCACCAC CGCCACAGCC
481 ATTAATGCAT CTTCCAAGGA GGAGTATGTG GTTTGCAGGA TATTCCATAA GAGCACTGGA
541 CTCAAGAAGG TAGTGATGTC GTCGTACGCC ATTCCCATGC CAATGTCCAT GGGAGCAGAG
601 GAGCAACATG GATTCCTTGA ATCCGGTACA TTGCCTCCTT TCATGGGCTA TGGTGCATCA
661 TCATCGCTGG TGCCCCCATC GTCCCTGCCC GCATCTTCTT CGTACCAGCT GCATGACGCT
721 GGGGCCAGAT CGTCAATGAT GGGTAGTGCG GTGCTCCCGA TGATGAACGA CCACTACTTC
781 GGGAACCACC ACTACCAGAA TATGGCTACC CCACCACGAC CGTTGGTGTC GTTCTACCAC
841 CATGACCCCC GCCAGCAGCA ACAACAACAA CAACACATGA TTCAAATGCC GATGCAGATG
901 CAGATAGGTG CAGATGAGGG CCTCATGATC GGGGTCGAGC CTGGGAGCGG GCCGTCATCC
961 ATAGTGTCGC AGGAGGACAC TTTGGCAAGG TTGAGTAGCA ACGACGTTGC AACAACTGCT
1021 GATGAGATCT CGTCAGTGAA CATGGGTACG GATGGCATGT GGAAGTACTG A
<210>5
<211>20
<212>DNA
<213> artificially synthesized sequence
<400>5
ATGGCAGACCACCTTCAAGT
<210>6
<211>22
<212>DNA
<213> artificially synthesized sequence
<400>6
TCAGTACTTCCACATGCCATCC
<210>7
<211>10
<212>PRT
<400>7
FGNHHYQNMATPPR
<210>8
<211>21
<212>DNA
<213> artificially synthesized sequence
<400>8
GCCTAGCCAATCTTCACAATC
<210>9
<211>20
<212>DNA
<213> artificially synthesized sequence
<400>9
TCAAGATCAAGCACGCCTAC
Claims (10)
1. The transcription factor for improving the gene expression of Seed Storage Protein (SSP) in the Wularg wheat is characterized in that: the transcription factor isTuNAC1The gene consists of a nucleotide sequence shown in SEQ ID No. 1; or isTuNAC1Homologous gene of gene in common wheatTaNAC1Three copies of (a):TaNAC-A1、TaNAC-B1andTaNAC-D1consists of nucleotide sequences shown in SEQ ID No.2, SEQ ID No.3 and SEQ ID No.4 respectively.
2. Comprising the transcription factor of claim 1TuNAC1The expression vector of (1).
3. Comprising the transcription factor of claim 1TuNAC1The wheat protoplast transient transformation system.
4. Prokaryotic expression of the transcription factor of claim 1TuNAC1The method of (2), characterized by: the transcription factor isTuNAC1The cells are connected and integrated into a prokaryotic expression vector and transformed into prokaryotic cells.
5. The method of claim 4, wherein the expression vector is a pGEX-4T-1 vector and the prokaryotic cell is E.
6. Overexpression of the transcription factor of claim 1 in endospermTuNAC1The method of (2), characterized by: and (3) transforming the endosperm of the common wheat by using the overexpression vector containing the transcription factor, preferably transforming the endosperm 15 d after the common wheat blossom by using a gene gun method.
7. Amplification of the transcription factor of claim 1TuNAC1The primer set of (2), characterized in that: the sequence of the forward primer is shown as SEQ ID No.5, and the sequence of the reverse primer is shown as SEQ ID No. 6.
8. The transcription factor of claim 1TuNAC1Use in a plant to increase SSP gene expression, characterized in that: the transcription factor isTuNAC1Over-expression in plants and screeningTuNAC1An overexpressed transgenic positive plant; preferably, the plant is Arabidopsis thaliana by containing the transcription factorTuNAC1Transforming Arabidopsis thaliana with the overexpression vector of (4)TuNAC1Overexpression of the gene.
9. The transcription factor of claim 1TaNAC1Use in a plant to inhibit SSP gene expression, characterized in that: transcription factors described in plantsTaNAC1Silencing and screening outTaNAC1Transgenic positive plants with the gene suppressed and expressed.
10. The use according to claim 11, wherein said plant is Triticum aestivum produced by comprising said transcription factorTaNAC1The RNAi vector is used for transforming common wheat to realizeTaNAC1Silencing of the gene.
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